High density lipoproteins (HDL) are attributed with an anti-atherogenic character, derived primarily from their facilitation of the reverse cholesterol transport (RCT) pathway. RCT plays a central role in lipid metabolism and cholesterol homeostasis by transporting lipids and cholesterol from peripheral tissues to the liver and steroidogenic tissues. Critical to RCT is the conversion of HDL associated cholesterol to cholesterylester by the enzyme lecithin:cholesterol acyltransferase (LCAT), resulting in the packaging of cholesterol at the HDL surface into the HDL core. This packaging leads to an ultrastructural transition of HDL from a discoidal to spherical shape. LCAT activity is dependent on interaction with the primary protein component of HDL, apolipoprotein A-I (apoA-I), which, like other exchangeable apolipoproteins, associates with lipid by modulating the exposure of its hydrophobic residues through conformational adaptation. The ability of apoA-I to foster LCAT activity is dependent on the extent of apoA-I's lipid association and its ensuing conformational state. Despite its significance on RCT and lipid metabolism, the molecular details underlying apoA-I's lipid-induced conformational adaptation, its regulation, and its functional consequences are not well understood. To determine the molecular mechanisms underlying this process, detailed structural knowledge of lipid-free and lipid-bound apoA-I must be obtained. We hypothesize that residues in the central "hinge" domain of apoA-I, necessary for LCAT activation, must adopt a particular lipid-dependent conformation for apoA-I to possess full LCAT activating capability. We propose to investigate the lipid-free and lipid-bound structure of apoA-I's central amino acids (82-163), by employing fluorescence and electron resonance techniques. Specifically, apoA-I's structure will be examined by site directed electron paramagnetic resonance spectroscopy (SDSL EPR) and fluorescence resonance energy transfer (FRET) in conjunction with circular dichroism (CD). Structural data will be correlated with LCAT binding and activation through surface plasmon resonance spectroscopy (SPR). This information will allow us to generate a structural context for apoA-I function and outline the molecular mechanism for the conformational adaptations that occur during lipid-association, its regulation and function.